&lt;P&gt;&lt;i&gt;&lt;b&gt;Structure and Function of Glycosyltransferases: &lt;/i&gt;&lt;/b&gt;To date, the detailed structure-function studies on glycosyltransferases, in particular on beta1,4-galactosyltransferase-1 (b4Gal-T1) from our laboratory, have shown following:&lt;/p&gt;&lt;P&gt;&lt;i&gt;&lt;b&gt; (I) Glycosyltransferases have flexible loop(s) in the vicinity of their catalytic pocket which undergo conformational changes upon donor substrate binding and create the acceptor binding site: (II) In the metal-ion dependent enzymes, the metal ion binding site is generally at the amino terminal hinge region of the flexible loop: (III) Glycosyltransferases interact with the add-on domains: &lt;/i&gt;&lt;/b&gt;To diversify the catalytic activity towards less preferred substrates, such as sugar acceptors or proteins or lipids or aglycons, the catalytic domains of glycosyltransferases either interact (1) with an additional protein, or have acquired add-on domains at the C-terminus or acquired add-on domains at the N-terminus. For example, in the lactose synthase enzyme, the b4Gal-T1, after conformational changes in the flexible loops to a closed conformation, interacts with a mammary gland-specific protein, alpha-lactalbumin (LA) at its carboxyl terminal end, changing the acceptor specificity of the enzyme towards less preferred acceptor glucose. LA protein, although not linked to b4Gal-T1, acts as an add-on domain. Several other glycosyltransferases have been shown or suggested to require an activating protein. In contrast to two interacting proteins, the catalytic domains of polypeptide a-N-Acetylgalactosaminyltransferases (ppGalNAc-Ts) have a lectin domain that is linked to at the C-terminus of the catalytic domain via a linker region and determines the specificity towards a peptide or a glycopeptide. The loops in the catalytic domain of these enzymes also undergo a conformational change upon binding of the metal ion and the sugar donor, while the lectin domain moves, bringing in the bound glycopeptide acceptor in the catalytic pocket, in order to synthesize O-a-GalNAc moiety on the glycopeptide. Also in this category is the alpha-1,6-Fucosyltransferase (FUT8), where an SH3 domain has been identified that is linked at the C-terminus of the catalytic domain.&lt;/p&gt; &lt;P&gt;&lt;i&gt;&lt;b&gt;(IV) A few residues in the catalytic pocket determine the donor sugar specificity of glycosyltransferases: Role of a single amino acid in the evolutionary divergence of invertebrate and vertebrate glycoconjugates: (a) Mutations in catalytic pocket of b4Gal-T1 change its donor specificity: &lt;/i&gt;&lt;/b&gt;Based on the structural information, we have previously shown, that the residue Tyr/Phe289 in the catalytic pocket of b4Gal-T1, which is conserved among all vertebrate homologs, when mutated to Leu or Ile broadens the donor substrate specificity of the enzyme to 2substituants of galactose i.e., GalNAc or 2-keto-galactose or 2-azido-galactose. (see Project # Z01 BC 010742). In invertebrates in the b4Gal-T homologs there is an Ile residue at the corresponding position of Tyr and they are b4GalNAc-T enzymes. Mutation of the Ile residue to Tyr in Drosophila b4GalNAc-T1 converts the enzyme to a b4Gal-T1 by reducing its N-acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold.&lt;i&gt;&lt;b&gt;(b) Few mutations in the catalytic domain of bovine alpha-1,3-galactosyltransferase (a3Gal-T) broadens the donor specificity: &lt;/i&gt;&lt;/b&gt;We have mutated bovine a1,3-galactosyltransferse (a3Gal-T) enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives by mutating the sugar donor-binding residues at positions 280 to 282. A mutation of His280 to Leu/Thr/Ser/Ala or Gly and Ala281 and Ala282 to Gly resulted in the GalNAc transferase activity by the mutant a3Gal-T enzymes to 5-19% of their original Gal-T activity. We show that the mutants 280SGG282 and 280AGG282 with the highest GalNAc-T activity can also transfer modified sugars such as 2-keto-galactose or GalNAz from their respective UDP-sugar derivatives to LacNAc moiety present at the nonreducing end of glycans of glycoprotein, thus enabling the detection of LacNAc moiety by a chemiluminescence method. This makes it possible to use these mutants, (1) for the detection of alterations in the glycosylation patterns in many pathological states, such as cancers and rheumatoid arthritis, and (2) in the glycoconjugation and assembly of nano-particles for the targeted drug delivery of bioactive-agents.&lt;i&gt;&lt;b&gt;(V) The N-acetyl group of the donor sugar is generally embedded in a hydrophobic pocket of the enzyme.&lt;/i&gt;&lt;/b&gt;In both mutant enzymes,Y289L-b4Gal-T1 and SGG-a3Gal-T, the N-acetyl moiety of the donor sugar GalNAc, is embedded in a hydrophobic pocket that allows the substitution of this moiety by CH2-CO-CH3 group. This acts as a chemical handle allowing conjugating with an amino-oxy group of a linking molecule.&lt;/p&gt;&lt;P&gt;&lt;i&gt;&lt;b&gt; Galectin -1 as a fusion partner for the production of soluble and folded beta-1, 4- Galactosyltransferase-T7 in E. coli: &lt;/i&gt;&lt;/b&gt;The expression of recombinant proteins in soluble and active form in E. coli often leads to aggregated proteins known as inclusion bodies. Modifying the bacterial growth conditions can sometimes solve the aggregation problem. Although we have developed an in vitro folding procedure that in many cases helps to fold the proteins from inclusion bodies, e.g., b4Gal-T1 or ppGalNAc-Ts, it nevertheless does not work with all the proteins. To date, the best available tool has been the use of several different fusion tags including the carbohydrate-binding protein, MBP that enhance the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. Here we show for the first time that another carbohydrate-binding protein galectin-1 can function as a fusion partner to produce soluble folded recombinant protein in E. coli. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for galectin-1, and a multi-cloning site where a cloned gene is inserted. The unique protease cleavage site allows the protein of interest to be cleaved from galectin-1 after lactose affinity column purification. Here we show that human beta1, 4-Galactosyltransferase-T7 (beta 4Gal-T7) fused to galectin-1 is produced as soluble, folded enzymatically active protein in E. coli. &lt;/p&gt; &lt;P&gt;&lt;i&gt;&lt;b&gt;Crystal structure of the catalytic domain of Drosophila &amp;#946;-1,4-galactosyltransferase-T7:&lt;/i&gt;&lt;/b&gt;Among the seven members of b4Gal-T family, the b4Gal-T7 transfers Gal from UDP-Gal to an acceptor, beta-xylose (bXyl), which is attached to side chain hydroxyl group of the Ser/Thr residue of proteoglycans, synthesizing a Gal-beta-1-4Xyl disaccharide moiety. Gene knockout studies in Drosophila have shown that b4Gal-T7 is essential for species survival while lack of b4Gal-T1 gene led to multiple disorders. However, mutations in the human b4Gal-T7 are known to cause skin fibroblasts of an Ehlers-Danlos syndrome. The catalytic domain of human b4Gal-T7 exhibits a 39% amino acid sequence similarity with the catalytic domain of human b4Gal-T1, while it shows a 68% sequence similarity with the catalytic domain of b4Gal- [summary truncated at 7800 characters]